ABSTRACT
Background: Insulin resistance is a frequent feature of chronic hepatitis
C. Whether insulin resistance could be the cause or consequence of steatosis
and fibrosis is unknown. The ability of HCV genotype 3 to promote steatosis
by itself provides an unique opportunity to answer this question.

Aims: The aim of the present study was to assess the relationships
between insulin resistance, steatosis, and fibrosis according to genotype
in 141 non-diabetic patients with biopsy proven non-cirrhotic chronic hepatitis
C.

Conclusion: This study shows that insulin resistance is the cause
rather than the consequence of steatosis and fibrosis in genotype 1 patients
and that increased circulating insulin is a risk factor for fibrosis through
insulin resistance induced steatosis.

BACKGROUND
Liver steatosis is common in chronic hepatitis C virus infection (HCV).1-3
Evidence supporting a detrimental role for steatosis in liver fibrosis progression
in chronic hepatitis C has been provided recently. Several studies have
shown that either fat accumulation in the liver or worsening are strong
and independent predictors of the severity and progression of fibrosis in
chronic hepatitis C infection, irrespective of the viral genotype involved.4,5,6,7,8,9,10
Two main mechanisms have been proposed to account for the high prevalence
of steatosis in chronic hepatitis C. Firstly, in patients infected with
genotype 3, the degree of steatosis is correlated with the level of viral
load,4,11 suggesting that HCV could alter fatty acid metabolism and/or export
in hepatocytes.12,13 Secondly, type 2 diabetes and more generally insulin
resistance is highly frequent in chronic HCV infection, as established by
several recent epidemiological studies.14-21 Whether or not insulin resistance
is a player in the development of the histological lesions of chronic hepatitis
C-as in alcoholic or non-alcoholic steatohepatitis-or simply the consequence
of viral infection and liver injury is debated. Recently, Shintani et al
have shown that insulin resistance preceded the occurrence of steatosis
in transgenic mice expressing HCV core protein, suggesting that insulin
resistance is not a consequence of hepatic steatosis in these mice.22 This
issue is of obvious practical importance because appropriate treatment should
be based on the pathophysiological process involved in the disease. In the
present study we addressed the following questions: is insulin resistance
the cause or the consequence of steatosis and fibrosis in chronic hepatitis
C? In other words, is insulin resistance, at least in part, responsible
for the occurrence and worsening of steatosis and fibrosis? What are the
determinants of insulin resistance in chronic hepatitis C? The unique ability
of HCV genotype 3 to promote by itself steatosis provides the opportunity
to clarify and unravel the complex relationships between insulin resistance,
steatosis, and fibrosis.

DISCUSSION
This study confirms that the pathophysiology of fatty liver associated chronic
hepatitis C is different in patients infected with genotype 1 and 3. The
study also shows that insulin resistance in patients infected with genotype
1 is the cause rather than the consequence of hepatic steatosis and fibrosis,
and suggests that increased circulating insulin is a risk factor for fibrosis
through insulin resistance induced steatosis.

Although steatosis was more severe in patients infected with genotype 3,
insulin resistance was associated with steatosis only in patients infected
with genotype 1. Insulin resistance depends mainly on age and is a major
risk factor for steatosis, independent of BMI. Steatosis was associated
with more severe fibrosis, whatever the genotype, supporting the major role
of steatosis, whatever its cause, in the progression of fibrosis. Indeed,
while univariate analysis identified a significant link between circulating
insulin level and fibrosis stage, multivariate analysis revealed that steatosis,
but not insulin, was independently associated with fibrosis, suggesting
an indirect effect of the circulating insulin level on fibrosis stage through
a steatosis related pathway.

To clarify the intricate relationship between insulin resistance, steatosis,
and fibrosis, the study was performed in two groups of patients with chronic
HCV infection due to genotype 1 or genotype 3. The results of the present
study confirm the cogency of this distinction as two distinct mechanisms
appear to operate in HCV associated fatty liver. In genotype 1 infected
patients, steatosis was linked to BMI, while in genotype 3 infected patients,
steatosis was related to HCV viral load. Moreover, we took care to exclude
patients with usual causes of steatosis, such as alcohol, drugs, or diabetes
mellitus. In this selected population, the prevalence of significant steatosis
(>=10%) was still high, reaching 60% in patients infected with genotype
3. These results are in accordance with previous studies showing a strong
association between steatosis and genotype 3 infection.4,5,6,7,8,9,10,11,12
Because cirrhosis is a well known cause of insulin resistance,31 patients
with biopsy proven cirrhosis were also excluded.

The mechanisms of development of insulin resistance in patients with chronic
HCV infection are not well understood. It has been suggested that insulin
resistance may result from steatosis, as excess free fatty acids could downregulate
insulin receptor substrate 1 (IRS-1) signalling.32 This concept was further
supported by recent evidence that reversing hepatic steatosis may improve
insulin resistance in rats with diet induced fatty liver.33 Despite the
significant relationship in genotype 1 infected patients, the lack of association
between steatosis and insulin resistance in genotype 3 infected patients
does not support this hypothesis. Shintani et al showed that insulin resistance
preceded the occurrence of steatosis in transgenic mice expressing HCV core
protein, suggesting that insulin resistance is not a consequence of hepatic
steatosis in these mice.22 As expected, BMI was correlated with the degree
of insulin resistance in univariate analysis.34 However, in multivariate
analysis, the degree of insulin resistance depended mainly on the age of
the patient. Finally, genotype 1 infected patients with fatty liver were
more insulin resistant than genotype 3 infected patients, probably because
they were older and had higher BMI values. It has been suggested that age
associated decline in mitochondrial function could contribute to insulin
resistance.35 Our results do not exclude other mechanisms. The higher prevalence
of type 2 diabetes in patients with chronic hepatitis C suggests implementation
of HCV infection itself.14-20 Impaired IRS-1 signalling could be a possible
mechanism, as recently shown in non-obese/non-diabetic patients with chronic
HCV infection.36 In a large cohort study, Hui et al showed a genotype specific
association between chronic HCV infection and insulin resistance.21 The
significant link between genotype 1 related steatosis and insulin resistance
in our study population tends to support this hypothesis.

As in previous studies, we found that steatosis (>=10%) was common among
patients with chronic hepatitis C, occurring in 34% of biopsy specimens.1-3
Our data confirm the strong correlation between the degree of steatosis
and level of HCV viraemia in genotype 3 infected patients.4 In patients
infected with genotype 1, multivariate analysis demonstrated that insulin
resistance was a risk factor for steatosis, independent of BMI. These data
confirm the existence of two distinct entities: a group of patients infected
with genotype 1 that may have steatosis secondary to metabolic causes such
as insulin resistance, and a second group infected with genotype 3 that
may have steatosis as a direct consequence of HCV infection.

In patients with chronic hepatitis C, we and others have shown a significant
relationship between the degree of steatosis and severity of fibrosis.4,6,7
In the present study, steatosis was associated with fibrosis, irrespective
of viral genotype. Because steatosis was associated with insulin resistance
in genotype 1 infected patients, fibrosis could be the result of hyperinsulinaemia.
In fact, it has been demonstrated that high levels of insulin and glucose
could promote fibrogenesis by stimulating the release of connective tissue
growth factor, a fibrogenic growth factor, from hepatic stellate cells.37
In genotype 1 infected patients, fibrosis stage significantly correlated
with circulating insulin levels in univariate analysis. When considering
other potential risk factors of fibrosis, such as steatosis, insulin was
no longer an independent risk factor. The relationship between steatosis
and fibrosis could be explained by several other mechanisms, such as lipid
peroxidation.38-41 According to the "two hits hypothesis", steatosis
could increase the sensitivity of hepatocytes to oxidative stress, the second
hit being HCV infection itself in patients with chronic hepatitis C.39 Production
of reactive oxygen species in an in vitro model expressing HCV core protein
is consistent with this hypothesis.42 The second independent risk factor
for fibrosis in the present study was the histological score of activity.
This result agrees well with the findings of previous longitudinal studies
that showed that the necroinflammatory score was predictive of the development
of severe fibrosis in patients with chronic hepatitis C.43,44 In our study,
neither the duration of contamination nor alcohol consumption was associated
with fibrosis. The non-linear progression of fibrosis in chronic hepatitis
C probably explains the former result.43,44 In most of the studies showing
a significant association between alcohol and fibrosis, the cut off value
was 20 g/day.5-7 In our study population, drinking less than 20 g/day, we
did not find any significant effect of small amounts of alcohol on fibrosis.

In conclusion, increased circulating insulin is a risk factor for fibrosis
in genotype 1 infected patients with chronic hepatitis C through insulin
resistance induced steatosis. Accordingly, it may be speculated that intervention
strategies to reduce insulin resistance associated with steatosis should
target these patients. In the near future, metformin or peroxisome proliferator
activated receptor {gamma} agonists could be interesting therapeutic options
for improving steatosis and fibrosis in HCV patients with insulin resistance.45,46

RESULTS
Characteristics of genotype 1 and genotype 3 infected patients A total of
141 patients met the inclusion criteria. The distribution of HCV genotypes
was as follows: 78 patients were infected by genotype 1, eight by genotype
2, 28 by genotype 3, 16 by genotype 4, four by genotype 5, one by genotype
6, and six had an undetermined genotype. The 113 patients infected with
genotypes other than 3 were collected into a single group termed "genotype
1 patients" and compared with the 28 patients infected with genotype
3. Their characteristics are listed in table 1. Both steatosis and fibrosis
were more frequent and more severe in patients infected with genotype 3
than in those infected with genotype 1. Other characteristics, in particular
those with a potential impact on both steatosis and fibrosis (namely, age,
disease duration, alcohol consumption, BMI, serum ALT activity, HOMA index
and histological activity score), did not differ between the two groups.

Insulin resistance is associated with genotype 1 related steatosis but not
with genotype 3 related steatosis Table 2 shows the main features of patients
infected with genotypes 1 and 3 according to the presence or absence of
significant steatosis. Genotype 1 patients with a fatty liver were older,
had a higher BMI, and were more frequently insulin resistant than those
without a fatty liver or those infected with genotype 3, even with marked
steatosis. These latter patients had a higher median viral load than that
found in the other groups. HOMA IR distribution in patients is presented
in fig 1. Median HOMA IR was significantly higher in patients with genotype
1 related steatosis than in those with genotype 3 related steatosis or patients
without steatosis. In genotype 1 patients, univariate analysis showed four
parameters significantly linked with the degree of steatosis: age, BMI,
HOMA IR, and hepatic iron concentration. Multivariate analysis showed that
HOMA IR was the only variable independently related to fatty liver in genotype
1 infected patients while in patients infected with genotype 3, viral load
was the only variable associated with fatty liver (r = 0.64, p = 0.003).

Insulin resistance is specifically associated with steatosis but not with
fibrosis in genotype 1 infected patients Genotype 1 infected patients with
extensive fibrosis (histological score >1) had a higher median HOMA IR
than others (1.8 v 1.0; p = 0.0001). However, the association was no longer
significant after adjustment for the degree of steatosis. Serum C peptide/insulin
ratio was similar in patients with or without extensive fibrosis (0.1 (0.03-0.6)
v 0.1 (0.03-0.4); p = 0.8).

Risk factors for insulin resistance in patients infected with genotype 1
In univariate analysis, seven parameters were significantly correlated with
HOMA IR in patients infected with genotype 1: age at liver biopsy, disease
duration, BMI, ALT activity, activity score, fibrosis score, and percentage
of steatosis. In multivariate analysis (r2 = 0.28), only age and degree
of steatosis were independently linked to HOMA IR.